Recently, two human primary cancers, leukemia and breast, and several human cancer cell lines, such as central nervous system, gastrointestinal tumors, and retinoblastoma, were shown to possess “side population” (SP) cells that have been described as cancer stem cells (1-5). Cancer stem cells, like somatic stem cells, are thought to be capable of unlimited self-renewal and proliferation. Multipotent cancer stem cells may explain the histologic heterogeneity often found in tumors (6-9).
In addition, cancer progression and metastasis may involve tumor stem cell escape from innate somatic niche regulators. Quiescent somatic stem cells residing in specific tissue niches until activation by injury or other stimuli have been described in skin and hair follicles, mammary glands, intestines, and other organs (10). The evolving evidence that somatic stem cells contribute to normal tissue repair and regeneration suggests the potential for multipotent somatic stem cells in the ovary responsible for regulated surface epithelial repair after ovulatory rupture and possibly the generation of oocyte nurse cells for folliculogenesis (11). Ovarian somatic stem cells would be expected to divide asymmetrically, yielding both a daughter cell that proceeds to terminal differentiation for epithelial repair and an undifferentiated self-copy. Repeated asymmetric self-renewal sets the stage for somatic stem cells or their immediate progenitors to accrue mutations over time, which might ultimately lead to their transformation into cancer stem cells and malignant progression.
Epithelial ovarian cancer, thought to emanate from the surface epithelium of the ovary (12, 13), consists of various histologic subtypes of Mullerian origin (serous, mucinous, and endometrioid), affects >22,000 women in North America per year, and accounts for >16,000 deaths per year with a projected 5 year mortality rate exceeding 70% (14). Aggressive surgical cytoreduction followed by chemotherapy results in complete clinical response in 50-80% of patients with stage III and IV disease. However, the majority of patients will relapse and become drug-resistant (16).
Various types of membrane-spanning ATP-binding cassette transporters, such as the multidrug-resistant gene 1 and breast cancer-resistance protein 1 (BCRP1), contribute to the drug resistance of many cancers, by pumping lipophilic drugs out of the cell (17). Within bone marrow, researchers have defined a subset of verapamil-sensitive BCRP1-expressing cells with the ability to efflux the lipophilic dye Hoechst 33342. This subset has been described as the side population (SP) (18).
Like somatic stem cells, cancer stem cells have the properties of self-renewal, heterologous descendent cells, slow cell-cycle times, and, unlike somatic stem cells, enriched tumor formation (8, 24).
Ovarian cancer patients initially respond well to surgical cytoreduction and chemotherapy. Chemotherapy alone can yield several logs of tumor cytoreduction but seldom a cure. The majority of patients who respond to primary chemotherapy ultimately develop recurrent, usually drug-resistant, disease that is likely due to the ability of ovarian cancer stem cells to escape these drugs.
The ovarian coelomic epithelium covers the ovary as a layer of simple squamous or cuboidal cells. In early embryonic development, the portion of the coelomic epithelium that covers the lateral tip of the urogenital ridge invaginates, while simultaneously undergoing an epithelial to mesenchymal transition (Zhan et al., 2006), giving rise to the mesenchyme of the Mullerian ducts (the anlagen of the oviduct, endometrium, and endocervix). This close embryonic relationship between ovarian coelomic epithelium and Mullerian structures is thought to explain the acquisition of Mullerian architecture and function during neoplastic progression.
Folliculogenesis in the adult ovary is characterized by extensive architectural remodeling that culminates in disruption of the coelomic epithelium and extrusion of the ovum at ovulation (Bjersing and Cajander, 1974, 1975; Bukovsky et al., 2004). After disruption, a subsequent series of molecular events initiates and executes repair of the epithelial wound (Clow et al., 2002; Tan and Fleming, 2004) by either non-stem mediated epithelial cell self-duplication or by stem cell mediated asymmetric division. Asymmetric somatic stem cell division has been proposed as a mechanism by which mutations may be accumulated and perpetuated to future generations (Reya et al., 2001) leading to neoplastic transformation. Concomitantly, previous studies of the coelomic epithelium implicating cyclic re-epithelialization as the source of accrued mutations leading to ovarian cancer (Murdoch et al., 2001) raise the possibility that an asymmetrically dividing coelomic epithelial somatic stem cell can have the propensity for transformation into an ovarian cancer stem cell.
Somatic stem cells are a subset of normal tissue cells that, through asymmetric division, have the ability to self renew and produce lineage committed daughter cells responsible for tissue regeneration and repair (Li and Xie, 2005). Such injury responsive somatic (non-hematopoietic) stem cells and their niches have been described in skin and hair follicle (Blanpain et al., 2004; Fuchs et al., 2004; Lowry et al., 2005; Tumbar et al., 2004), mammary gland (Welm et al., 2003; Welm et al., 2002), intestine (Leedham et al., 2005; Li and Xie, 2005; Mills and Gordon, 2001; Spradling et al., 2001; Vidrich et al., 2003; Williams et al., 1992; Wong, 2004), and other tissues (Alvarez-Buylla and Lim, 2004; Berns, 2005; Imitola et al., 2004; Liu et al., 2004). In some tissues, slow cycling somatic stem cells were initially identified by their ability to retain labels for long periods of time, while asymmetrically derived lineage committed daughter cells dilute out the label during rapid proliferation and terminal differentiation (Albert et al., 2001; Braun and Watt, 2004; Kenney et al., 2001; Morris et al., 1986; Morris and Potten, 1994, 1999; Oliver et al., 2004; Tsujimura et al., 2002; Tumbar et al., 2004; Watt and Hogan, 2000; Wu and Morris, 2005). These studies, as well as those leading to the recent identification of LRCs in the uterine endometrial stroma and myometrium (Szotek et al., 2007), used BrdU (or 3H-Thymidine) or histone H2Bj-GFP labeling to identify and isolate somatic stem cells which, in turn, permitted the discovery of tissue specific surface markers.
Concurrently, Goodell et al. devised another method to isolate previously unidentified stem or progenitor cells by demonstrating that hematopoietic and mammary gland stem cells have the ability to efflux Hoechst 33342 dye through ATP-binding-cassette (ABC) transporters such as Abcg2/Bcrp1 (Goodell et al., 1996; Welm et al., 2002). These “side population” (SP) cells have since been correlated with somatic and cancer stem cells from various tissues (Al-Hajj et al., 2003; Behbod and Rosen, 2005; Bhatt et al., 2003; Haraguchi et al., 2005; Jonker et al., 2005; Kondo et al., 2003; Patrawala et al., 2005; Preffer et al., 2002; Smalley and Clarke, 2005; Smalley et al., 2005; Wulf et al., 2001), including our recent identification of SP cells in ovarian cancer populations (Szotek et al., 2006). Thus, label retention and Hoechst dye efflux are two distinct methods based on stem cell functional properties that can be used individually or potentially in combination to identify candidate somatic stem cells without known surface markers and to define a signature stem cell marker profile.